The invention concerns a method of inclusion of microorganisms of the group consisting of mycorrhizae and actinorhizae in a matrix comprising a polymer gel, to prepare a product suitable for use in particular for agronomic purposes.
It was proposed many years ago to fix microorganisms on a carrier in U.S. Pat. No. 1,909,622, in which nitrogenfixing bacteria of the genus Rhizobium are used.
Generally, however, the microorganism culture is adsorbed onto a carrier, as in Belgian patent 521,850, which advocates the use of diatomaceous earth and colloidal silica for Rhizobium. Bentonite has also been used (in British Patent No. 1,777,077), as have plaster granules (in French Patent No. 1,490,046) and even lignite.
This method of inclusion by adsorption has disadvantages, particularly in connection with the survival of the microorganism and its protection during transport, storage and handling. Since this procedure gives only very limited results, attempts have been made to improve fixing techniques.
Thus, French Patent No. 1,180,000 calls for the use of a must juice, to which substances with an adsorbing action are added, such as cellulose, bone meal, kaolin or silica gel, in the manufacture of preparations rich in bacteria of the Azotobacter group. This type of preparation is also not satisfactory.
Attempts have therefore been made to improve both the survival rate of the microorganisms, e.g., by inclusion, and the manner of bringing them into the plant medium. Thus, in French Patent application No. 77.10254 of Apr. 5, 1977 (corresponding to U.S. Pat. No. 4,155,737) a method is proposed which makes use of an inoculum comprising a polymer gel with microorganism included therein. The inoculum is introduced into the rhizosphere of the plants. According to this patent, the polymer gel may be a polyacrylamide gel or a silica gel.
None of the above methods has been entirely satisfactory in meeting all of the requirements for an adequate carrier. Such a carrier must maintain the microorganism under conditions sufficient to preserve, protect and keep it in a suitable form for handling, while at the same time enabling it to swarm into the medium and if possible allowing for the grafting of additives onto the carrier. Furthermore, the carrier must insure the viability of the microorganism, even after periods of several weeks and under conditions of variable hygrometry. This means that the carrier must be able either to contain a sufficient reserve of water, which can be made available as necessary, or to obtain the necessary water from the environment. Finally, the carrier must not be detrimental to the environment, i.e., it must be either biodegradable or non-polluting.
This rapid enumeration of requirements, which makes no claim to be exhaustive, suggests why until now a suitable method has not been developed as expected.
In French Patent application 79.08597 of Apr. 5, 1979, there is disclosed a particularly attractive method, comprising the inclusion of the microorganism in a polymer of the polysaccharide group and an at least partial cross-linking of the polymer, e.g., by heat treatment, through use of a metal salt or through synergism with another polymer. This method gives surprising results, and a remarkable synergism is shown when a compound which is absorbent and adsorbent, such as a silica, is also used.
The above-noted French patent application is directed in particular to preparations of Rhizobium japonicum, a non-sporulated, nitrogen fixing bacterium which is very sensitive to drying, temperature and physico-chemical factors. Some microorganisms, however, present additional difficulties because of their fibrous nature, such as ectomycorrhizian fungi (e.g., Pisolithus, Hebeloma, Tuber, Boletus, etc.) which, moreover, have no form of resistance to, e.g., pathogenic germs, when cultivated in pure culture.
Mycorrhizian associations are the result of associating a fungus with a root, leading to a true symbiosis. The following groups are distinguished by the nature the association:
(a) ectotrophic mycorrhizae, found chiefly in woodland trees (Pinaceae, Fagaceae), most being upper fungi (Ascomycetes and Basidiomycetes); and
(b) endotrophic mycorrhizae, usually lower fungi (Phycomycetes), much more common than the above and found in trees, shrubs and herbaceous plants in the case of mycorrhizae with arbuscles and vesicles, but limited to Ericaceae in the case of mycorrhizae with clusters.
The beneficial action of these mycorrhizae on the growth of plants can be attributed to plant hygiene protection against pathogens in the ground, production of growth-promoting substances or vitamins improvement in the mineral nutrition of the plant (particularly with respect to phosphorous) through an increase in the possibilities for ground exploration, and improvement in water absorption where there is a shortage of water.
In the case of nitrogen-fixing non-leguminous plants, symbiotic associations of the same type as those existing between Rhizobium and leguminous plants are characterized by the formation of nodules on the root system. These are found both in trees and shrubs and in herbaceous plants. The function of the nodules is known only in certain ligneous plants which generally colonize poor or degraded soils (sands, moraines), where real fixation of atmospheric nitrogen has been shown to take place. 137 species of Angiospermae belonging to 12 different genera, classed in seven families (Betulaceae, Casuarinaceae, Coriariaceae, Eleagnaceae, Myricaceae, Rhamnaceae, Rosaceae) have been recognized (Bond 1974). The endophyte responsible for forming the nitrogen-fixing nodules is an Actinomycetum (Frankia) which had only been isolated in pure culture in 1978 by Lalonde et al. (Laval University, Quebec).
There is no doubt of the importance of using these nitrogen-fixing varieties of microorganisms in sylviculture, particularly in marginal soils which are poor in nitrogen and with a modified structure or none at all. The following examples can be given as an indication of typical uses:
(1) reforestation of peat bog in France (Aulne, Myrica), glacial moraines in the Alps (Aulne), soil thrown up in mining or quarrying oil shale in the U.S.A.;
(2) fixing maritime and continental dunes in Senegal (Casuarina);
(3) developing James Bay in Canada;
(4) Alnus rubra - Douglas associations in the N.W. American forest systems;
(5) use of Alnus glutinosa, A. cordata, A. incana, A. crispa as nurse trees to encourage development of nonfixing species;
(6) use of Ceanothus, Myrica, Hipophaca, Eleagnus in association with non-fixing species or in production of green fertilizer or biomass.
Inoculation has been traditionally carried out at the nursery stage, using crushed nodules. This has the serious disadvantages that pathogenic germs may be introduced into the inoculum and that the nodules cannot be preserved, even at low temperature, because of the rapid oxidation of tannins and phenolic substances, which are toxic to the microorganisms. It has not thus far been possible to demonstrate in a formal manner that the productivity of these species can be increased by inoculating with an endophyte developed in pure culture.
With ectomycorrhizae, inoculation has traditionally been carried out using soil from another nursery; currently, it is more common to use pure cultures of mycorrhizian fungi in vermiculite. Inoculation carried out in this way has three major disadvantages:
(1) the difficulty in developing the fungus (e.g., Pisolithus or Hebeloma): At least six weeks time is required (Grahan-Linderman 1980 Can. J. Microb. 26, II) to obtain a thin, heterogeneous culture in a liquid medium; at least eight to ten weeks is required on vermiculite. In the case of a liquid culture, it is necessary to recover the mycelium and crush it before use, with the dangers of excessive shearing leading to no regrowth. This is particularly true for Tuber, since the microorganism has only hyphae and no form of resistance.
(2) the large quantity of inoculum required: 2 l/m.sup.2 in the case of microorganisms developed on vermiculite, with the corresponding difficulty in storage; and
(3) the need to inoculate with inocula which have been freshly prepared or stored at 4.degree. C. It is these problems which have led to the development of the instant invention.
One object of the invention is to develop an inoculum which can be stored at ambient temperature and is easy to use.
Another object is to improve the properties of cultures of mycorrhizae and actinorhizae, particularly with respect to the level of homogeneity.
Still another object of the invention is to achieve a reduction of cultivation time, in particular for actomycorrhigae